LEOPARD syndrome (LS), a disorder with multiple developmental abnormalities, is mainly due to mutations that impair the activity of the tyrosine phosphatase SHP2 (PTPN11). How these alterations cause the disease remains unknown. We report here that fibroblasts isolated from LS patients displayed stronger epidermal growth factor (EGF)-induced phosphorylation of both AKT and glycogen synthase kinase 3␤ (GSK-3␤) than fibroblasts from control patients. Similar results were obtained in HEK293 cells expressing LS mutants of SHP2. We found that the GAB1/phosphoinositide 3-kinase (PI3K) complex was more abundant in fibroblasts from LS than control subjects and that both AKT and GSK-3␤ hyperphosphorylation were prevented by reducing GAB1 expression or by overexpressing a GAB1 mutant unable to bind to PI3K. Consistently, purified recombinant LS mutants failed to dephosphorylate GAB1 PI3K-binding sites. These mutants induced PI3K-dependent increase in cell size in a model of chicken embryo cardiac explants and in transcriptional activity of the atrial natriuretic factor (ANF) gene in neonate rat cardiomyocytes. In conclusion, SHP2 mutations causing LS facilitate EGF-induced PI3K/AKT/GSK-3␤ stimulation through impaired GAB1 dephosphorylation, resulting in deregulation of a novel signaling pathway that could be involved in LS pathology.Noonan syndrome (NS) (MIM163950) is a relatively frequent (ϳ1/2,000 births) autosomal dominant disease primarily characterized by facial dysmorphism, heart defects, and short stature. LEOPARD syndrome (LS) (MIM151100) is a rarer but related disorder that associates, roughly, NS symptoms with deafness and cutaneous abnormalities. Both NS and LS belong to the family of "neuro-cardio-facial-cutaneous" (NCFC) syndromes, a group of developmental disorders that display different combinations of the above-mentioned symptoms with mental retardation and tumor predisposition (4, 13).At least 80% of LS and 50% of NS patients carry germ line missense mutations in PTPN11, the gene encoding SHP2. SHP2 is a widely expressed protein tyrosine phosphatase (PTP) that contains Src homology 2 (SH2) domains and promotes Rasmitogen-activated protein kinase (MAPK) activation through different molecular mechanisms (14,27,39,47). Other LS or NS forms are caused by mutations in KRAS, SOS1, RAF1, BRAF, and SHOC2 genes, which encode key factors of the Ras-MAPK pathway (6,32,34,37,38,41).Although genetic studies have provided essential advances, how PTPN11 mutations cause the diseases' symptoms remains an open question. Biochemical studies have shown that NS mutations are located at contact points between the catalytic and the SH2 domains and therefore disrupt SHP2 autoinhibitory conformation (11,19), thereby stimulating SHP2 catalytic activity (gain-of-function mutations). Conversely, LS mutations are confined within the catalytic domain and repress SHP2 activity (15,21,40).A key issue in understanding LS/NS pathogenesis is the identification of the signaling pathways that are altered by these mutations. It is now well ac...

Somatic mutations in the calreticulin (CALR) gene are associated with approximately 30% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). CALR mutations, including the two most frequent 52 bp deletion (del52) and 5 bp insertion (ins5), induce a frameshift to the same alternative reading frame generating new C-terminal tails. In patients, del52 and ins5 induce two phenotypically distinct myeloproliferative neoplasms (MPNs). They are equally found in ET, but del52 is more frequent in PMF. We generated heterozygous and homozygous conditional inducible knock-in (KI) mice expressing a chimeric murine CALR del52 or ins5 with the human mutated C-terminal tail to investigate their pathogenic effects on hematopoiesis. Del52 induces greater phenotypic changes than ins5 including thrombocytosis, leukocytosis, splenomegaly, bone marrow hypocellularity, megakaryocytic lineage amplification, expansion and competitive advantage of the hematopoietic stem cell compartment. Homozygosity amplifies these features, suggesting a distinct contribution of homozygous clones to human MPNs. Moreover, homozygous del52 KI mice display features of a penetrant myelofibrosis-like disorder with extramedullary hematopoiesis linked to splenomegaly, megakaryocyte hyperplasia and the presence of reticulin fibers. Overall, modeling del52 and ins5 mutations in mice successfully recapitulates the differences in phenotypes observed in patients.